Glycated proteins are generated by non-enzymatic covalent bonding between aldehyde groups in aldoses, such as glucose (monosaccharides potentially containing aldehyde groups and derivatives thereof), and amino groups in proteins, followed by Amadori rearrangement. Examples of amino groups in proteins include α-amino group of amino terminus and side chain ε-amino groups of lysine residue in proteins. Examples of known glycated proteins generated in vivo include glycated hemoglobin resulting from glycation of hemoglobin and glycated albumin resulting from glycation of albumin in the blood.
Among such glycated proteins generated in vivo, glycated hemoglobin (HbAlc) has drawn attention as a glycemic control marker significant for diagnosis of diabetic patients and control of conditions in the field of clinical diagnosis of diabetes mellitus. The blood HbAlc level reflects the average blood glucose level for a given period of time in the past, and the measured value thereof serves as a significant indicator for diagnosis and control of diabetes conditions.
As a method for quickly and simply measuring HbAlc, an enzymatic method involving the use of amadoriases, wherein HbAlc is decomposed by a protease or other substance and α-fructosyl valyl histidine (hereafter, referred to as α-FVH) or α-fructosyl valine (hereafter, referred to as α-FV) released from the β-chain amino terminus is quantified, has been proposed (e.g., Patent Documents 1 to 6). According to a method in which α-FV is cleaved from HbAlc, in fact, the influence of contaminants is considered to be significant. At present, accordingly, a method in which α-FVH is measured is the major technique.
An amadoriase oxidizes iminodiacetic acid or a derivative thereof (also referred to as an “Amadori compound”) in the presence of oxygen to catalyze a reaction to generate glyoxylic acid or α-ketoaldehyde, amino acid or peptide, and hydrogen peroxide.
Amadoriases have been found in bacteria, yeast, and fungi. Examples of known amadoriases having enzyme activity to α-FVH and/or α-FV, which are particularly useful for measurement of HbA1c, include amadoriases derived from the genera Coniochaeta, Eupenicillium, Arthrinium, Curvularia, Leptosphaeria, Neocosmospora, Ophiobolus, Pleospora, Pyrenochaeta, Cryptococcus, Phaeosphaeria, Aspergillus, Ulocladium, and Penicillium (e.g., Patent Documents 1 and 7 to 11; Non-Patent Documents: 1 to 4). In some of the aforementioned documents, an amadoriase is occasionally referred to as, for example, ketoamine oxidase, fructosyl amino acid oxidase, fructosyl peptide oxidase, or fructosyl amine oxidase.
In the measurement of HbAlc by an enzymatic method, amadoriases are required to have stringent substrate specificity. When HbAlc is measured by quantifying released α-FVH as described above, for example, use of amadoriases that are less likely to react with glycated amino acids or glycated peptides other than α-FVH that are present freely in specimens and/or released in the process of HbAlc treatment using proteases or the like is preferable. In particular, side chain ε-amino groups of lysine residues contained in the hemoglobin molecules are known to undergo glycation, and ε-fructosyl lysine in which an amino group at position ε derived from the glycated lysine residue has been glycated (hereafter, referred to as “ε-FK”) is released by treatment with proteases or other substances (e.g., Non-Patent Document 5). Accordingly, amadoriases having high substrate specificity, which are less likely to react with ε-FK, potentially causing measurement errors, are strongly desired. However, the reactivity of most known amadoriases with ε-FK cannot be said to be sufficiently low.
As a general technique, a method of adding mutations to DNAs encoding enzymes, introducing substitutions into the amino acids of enzymes, and selecting enzymes with substrate specificity of interest in order to alter the substrate specificity of the enzymes is known. If an example of improving substrate specificity by amino acid substitution in enzymes with high homology is already known, further, improvement in the substrate specificity can be expected based on such information.
Regarding ketoamine oxidase derived from Curvularia clavata YH923 and ketoamine oxidase derived from Neocosmospora vasinfecta 474, in fact, modified ketoamine oxidase having altered substrate specificity for α-FVH resulting from substitution of several amino acids has been found (Patent Document 1). In the case of ketoamine oxidase derived from Curvularia clavata YH923, for example, substitution of isoleucine at position 58 with valine, arginine at position 62 with histidine, and phenylalanine at position 330 with leucine is found to reduce the ratio of activity (i.e., ε-FZK/α-FVH), which is determined by dividing enzyme activity to ε-fructosyl-(α-benzyloxycarbonyl lysine) (hereafter, referred to as “ε-FZK”) by enzyme activity to α-FVH to result in a figure from 0.95 to 0.025.
However, ε-FZK used for evaluation of substrate specificity of a modified ketoamine oxidase in the aforementioned document is very different from ε-FK that is actually generated in the process of treatment of glycated hemoglobin with a protease in terms of molecular weight and structure. Accordingly, it is difficult to conclude that reactivity to ε-FK, which could actually cause measurement errors, is reduced based on reduced reactivity to ε-FZK. In addition, there is no description to the effect that reduction in reactivity to ε-FK was confirmed with the use of the modified ketoamine oxidase in the aforementioned document.
In addition, modified fructosyl amino acid oxidase resulting from introduction of amino acid substitution into fructosyl amino acid oxidase derived from Aspergillus nidulans A89 to alter substrate specificity, thereby additionally imparting reactivity to α-FVH thereto, has been reported (e.g., Patent Document 10). For example, substitution of serine at position 59 with glycine and lysine at position 65 with glycine or substitution of lysine at position 109 with glutamine of fructosyl amino acid oxidase derived from Aspergillus nidulans A89 is found to additionally impart enzyme activity to α-FVH. However, there is no description to the effect that such amino acid substitution would contribute to a reduction in reactivity to ε-FK.
There is another report regarding a modified fructosyl amino acid oxidase derived from fructosyl amino acid oxidase derived from Aspergillus nidulans A89, which is obtained by amino acid substitution to alter substrate specificity, thereby reducing the ratio of activity (i.e., ε-FK/α-FV), which is determined by dividing enzyme activity to ε-FK by enzyme activity to α-FV (e.g., Patent Document 12). However, there is no description regarding the activity of such modified enzyme on α-FVH.
Including naturally-occurring and modified amadoriases, specifically, only a very small number of reports have been made regarding amadoriases having low the ratio of activity (i.e., ε-FK/α-FVH and/or ε-FK/α-FV), which is determined by dividing enzyme activity to ε-FK by enzyme activity to α-FVH. Accordingly, there continues to be a need for amadoriases having sufficiently low reactivity to ε-FK enabling accurate measurement of HbAlc.